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To both improve the efficiency of contact isolation among ESBL-PE carriers and avoid the unnecessary isolation of noncolonized patients, we should reduce the turnaround time of ESBL screening in laboratories and improve the sensitivity of diagnostic methods. The development of rapid and low-cost methods that satisfy these two goals is a promising approach.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify pathogens, despite some limitations of the technique. The Enterobacter cloacae complex (ECC) taxonomy has recently been expanded, leading to uncertain identification of some species within the ECC when commercial MALDI-TOF MS is used. This technique is especially unsuited in the case of E. hormaechei, the main species responsible for infections and one of the most prone, within the ECC, to acquire antibiotic resistance. Hence, rapid and reliable identification at the species level could improve patient management. Here, we evaluated the performance of the Bruker Microflex MALDI-TOF MS instrument to identify ECC isolates using two databases and algorithms in comparison to the hsp60 gene sequencing reference method: the Bruker database included in the MALDI Biotyper software and an extensive online database coupled to an original Mass Spectrometric Identification (MSI) algorithm. Among a panel of 94 ECC isolates tested in triplicate, the online database coupled to MSI software allowed the highest rate of identification at the species level (92%) compared to the MALDI Biotyper database (25%), especially for the species E. hormaechei (97% versus 20%). We show that by creating a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software, we were able to substantially improve the identification of the E. cloacae complex members, with only 8% of isolates misidentified at the species level. This online database is available through a free online MSI application (https://msi.happy-dev.fr/). IMPORTANCE Creation of a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software enables substantial improvement in identification of E. cloacae complex members. Moreover, this online database is available through a free online MSI application (https://msi.happy-dev.fr/).

An atypical Enterobacteriaceae strain with a β-lactam susceptibility pattern of inducible cephalosporinase was isolated in Tenon Hospital (Paris, France) from a patient’s skull wound infection. Identifications by the API-50CHE biochemical system and 16S rRNA gene sequencing concluded that it was a member of the Buttiauxella genus. The bla gene was cloned and sequenced. The deduced translated product was a 383-amino acid protein (BUT-1) with 75–78% identity with the chromosomal AmpC β-lactamases of Citrobacter freundii , Enterobacter aerogenes , Enterobacter cloacae and Escherichia coli . The isoelectric point of 9.0 and the kinetic constants of BUT-1 were comparable with results described for other Ambler class C enzymes. bla BUT-1 and the associated ampR transcriptional regulator gene were divergently transcribed from a common intercistronic region, a genetic organization already described for other inducible class C β-lactamases. The deduced amino acid sequence of AmpR shared 85% and 81% identity with AmpR from E. cloacae and C. freundii respectively.

Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates.

Eleven Klebsiella pneumoniae clinical isolates and one Klebsiella oxytoca clinical isolate showing various pulsed-field gel electrophoresis types and producing an inducible DHA-1 class C β-lactamase were isolated in the Parisian region between 1998 and 2003. The aim of this study was to compare the genetic organization of the bla DHA-1 genes in this collection of clinical isolates. In four isolates, the Morganella morganii -derived genomic region containing bla DHA-1 was inserted in an entire complex sul1 -type integron, including a region common to In6-In7 (CR1), as previously described in a bla DHA-1 -producing Salmonella enterica serovar Enteritidis KF92 isolate from Saudi Arabia in 1992. Different gene cassette arrays were characterized in each of these integrons. In two of them, an additional 10-kb fragment was inserted between the CR1 and the M. morganii -derived region and was similar to the sap (ABC transporter family) and psp (phage shock protein) operons originated from Salmonella enterica serovar Typhimurium. The length of the M. morganii region was variable, suggesting that several independent recombination events have occurred and that open reading frame orf513 encodes a recombinase involved in the mobilization of the resistance genes. The genetic organization of bla DHA-1 was identical in the eight other isolates. This structure is likely derived from a complex integron following the insertion of IS 26 , leading to the deletion of the first part of integron. The horizontal transfer of one plasmid carrying that truncated integron was shown for seven of these isolates.

The amplification and sequence of ampC genes in Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei and Enterobacter intermedius bring the number of known cephalosporinase sequences from the genus Enterobacter to seven. Expression in Escherichia coli of the ampC genes from E. asburiae, E. hormaechei and E. intermedius established the functional nature of these genes. ampC from E. asburiae shows 96.5% identity to bla(ACT-1) encoding a plasmid-borne cephalosporinase previously believed to derive from Enterobacter cloacae. The reassignment of ACT-1 ancestry to E. asburiae is confirmed by the 95.5% identity between ampR upstream of bla(ACT-1) and ampR from E. asburiae.